KR970000596B1 - In vitro embryo culture technique - Google Patents

Abstract

Avian embryos may be cultured in vitro up to blastoderm formation, during embryonic morphogenesis and/or during embryonic growth to hatch. The invention may have applications not only in the genetic engineering of poultry, but also in the investigation of fundamental mechanisms of avian development and in the study of deleterious traits. Moreover, it may afford a desirable alternative to surgical intervention in the laying hen.

Description

Test tube embryo culture

1 is a diagram of the culture system of a chick embryo showing the period of improvement protected by individual and connected systems.

2 is the structure of a new hen's egg.

3 is a diagram of the culture system of phase III (hatching 4 days).

4 is a schematic of phase II culture system (1-4 days or 9 days).

5 is a diagram of phase I culture system (ovil modified to day 1).

The present invention relates to a method of culturing in vitro bird embryos suitable for use in poultry, in particular hens.

In the initial stages of growth from fertilization to cell division, chick embryos could not be treated as experimental adjustments due to egg size, weakness and relatively difficult brittleness. These issues have been discussed in recent years as a possible way for exogenous generation to turn into algae. (Freeman and Messer, 1985; Crittenden and Salter, 1986). Perry (1986 a, b) has been conceived from a selective perspective as being able to carry out genetic modification of birds. The purpose of studying a complete culture system of chick embryos is to provide a means for growing adjusted eggs to maturity. The method is to take in vitro culture at an intermediate stage of embryo growth and development occurs because of this. This method is not only applied to genetic engineering of poultry, but also used to study the basic mechanisms of algae improvement and to study the hazards. Furthermore, surgical adjustments in oviparous hens can be chosen as desired.

The chick embryo is located in the cytoplasmic small part which is located in the betrayal and egg animal play (usually yolk masturbation). At one third of the growth, the vessel floats on the surface of the yolk and the outer-membrane grows around the yolk and a vasculature is made. During the rest of the growth, the pear grows by consuming the nutrients preserved in the eggs.

For this purpose, the growth of chicks is divided into three phases with varying requirements in successive stages from fertilization to hatching.

Phase I. Fertilization with germ formation. This statue takes place in the oviduct and ends in the egg position. Spouse interaction occurs within 15 minutes after ovulation and the first cell division occurs after about 4 hours (Ferry, 1987). Twenty hours later, the next division occurs in a single sheet of cells that spans the embryonic cavity (Kohhaus, Genesberg and Eal-Guiladi, 1980). During passage through the oviduct, the egg is given a large variety of whites from the isthmus where the cell division begins to the cornea. In the uterus, the egg white is doubled in volume by absorption of the uterine secretion (absorption distribution), and finally the angle gradually calcifies. In hens that lay one egg in succession for a long time each day, the egg position is achieved within 15-30 minutes by the next ovulation and the circulation is repeated.

Phase II. Pear form generation. This phase occurs on the first 3 days of hatching of eggs (steps 1-18, Hamburger and Hamilton [1951]).

In step 20, the embryo is 10 mm long and the germ of the outer embryo is expanded around the yolk to its equator.

Phase Ⅲ. Pear growth. This phase occurs on the last 18 days of egg hatching (steps 18-45, Hamburger and Hamilton [1951]).

Some methods are available for short term culturing of embryos in Phase II (New, 1960) and for long term cultivation of more advanced embryos (Dun, Fitzharis and Barnett, 1981; Ono and Wakkagi, 1984; Lowe). Let and Simkis, 1985, 1987). Some methods implant pears from yolks, while others implant pears and original yolks into culture vessels. The latter method provides more desirable conditions for long term culture and is uniquely used in current culture systems.

According to a first aspect of the present invention, there is provided a method for in vitro culture of algae embryos on embryo growth, which seals the embryos in a closed vessel, allows air to escape on the vessels, and allows the gas to partially permeate the vessels. It is separated from the outside atmosphere by means of a seal.

The seal is preferably in the form of a membrane. The seal is plastic, for example polyethylene. Commercially available mucosa forms suitable seals, especially when used in two layers. Any other material having suitable properties with the mucous membrane can be used.

The suitability of the seal material is determined indirectly by measuring carbon dioxide or water vapor permeability; Permeation of carbon dioxide can be measured by testing the rise of the pH of the front egg white after incubation at 38 ° C. for 24 hours in another permeable container.

The white egg is the first to inject gas into carbon dioxide, bringing the pH down to 0.1. It is generally suitable for the pH to rise to 0.5-1.5, preferably 0.5 or 0.7-1.0 or 1.3, for example 0.9. Water vapor permeability is 5 or 10-30 or 40 mg / cm 2 / 24h.

The container is preferably a portion of eggs which are usually selected from the same species being cultured. In particular, it can be seen that it is suitable to remove blunt ends from the whole egg; Particularly suitable is a 40 mm diameter hole in the center of the egg axis.

At the blunt end of the egg, the hole is partially sealed by a gas permeable seal. The seal is made to stick to the egg shell with whites. Preferably the permeability properties are similar to those of natural eggs.

The incubator is present with several features of the present invention, particularly when the conventional culturing step has been performed in vitro but without which the method cannot be performed, for example when the conventional culturing step is performed naturally. The incubator present usually consists of white undiluted or diluted form and the desired inter-uterine secretion.

When using hen eggs, the space depth between the vessel and the seal is 5-15 mm, for example about 10 mm.

At first it is desirable to disturb gently cultured embryos, and gentle disturbances can be achieved, for example, by intermittent shaking at a 30 ° angle. Typical incubation temperatures are maintained at, for example, about 38 ° C.

In vitro cultures in accordance with aspects of the present invention are suitable for use from about 4 days (calculated in fertilization) to about 25 days of hatching that normally occurs. However, during the last few days of embryonic life (eg 13 days), it is desirable that there is no disturbance. Furthermore, it is preferable to drive the seal so that a certain amount of air is injected into the container some time before the determined incubation time (for example, 1-2 days ago). Subsequently, additional air can be injected by optionally drilling the egg shell (when the eggs form a container) and removing the seal, for example by a solid disk fed by a petri dish.

According to a second aspect of the invention, there is provided a method for in vitro culture of algae embryos in embryonic form development, which means culturing the embryos in an incubator in a closed, mac-impermeable container filled with liquid.

The vessel can partially permeate the gas. Gas permeability is provided by egg shells (generally in combination with the inner cornea) or other partial gas permeable seals, the preferred properties of which constitute the first feature of the present invention. It can be seen that the egg shell and the inner cornea partially permeate gas.

The incubator is preferably liquid white collected from new eggs. The container is preferably a portion of eggs, but preferably the structure is different from that of the first feature of the invention. In this aspect, for example, a die which is removed by a 32 mm hole is preferable.

This ensures the presence of air chambers between the outer and inner corneas; This is beneficial because air chambers expand during incubation to recover the parts lost by evaporation.

If a seal is used to seal off the tip, the egg is usually incubated in a horizontal position because there is a seal on one side. The seal is held firmly against the shell of the egg.

It is highly desirable that cultured embryos be gently disturbed.

For example, it is desirable to shake intermittently or continuously at an angle of 80 ° at time intervals or at other comparable intervals.

Other methods according to the features of the present invention usually start about 1 day after fertilization (ie normal time to lay eggs) and continue until 2 days or more than 2, for example 8 days. However, it is preferred that the method according to the features of the invention continue for 3 or 4 days before transplanting the embryo by the method according to the first feature.

A third aspect of the present invention provides a method for in vitro culturing of algal embryos during embryonic morphogenesis and on embryo growth, which method is to culture embryos by the method according to the second aspect of the present invention.

The transplantation is preferably 2 to 5 days after fertilization, for example 4 days.

According to a fourth aspect of the invention, there is provided a method for in vitro culturing of embryonic embryos up to array formation, which means culturing the fertilized egg with the surrounding capsules of dark white partially precipitated in the incubator. For best results, the incubator should be on the line with the betrayal, which is usually the highest, but below the level of the white capsule.

Fertilized eggs are apparently obtained from hens. If surgical methods are used, eggs should be medium in size, eg 50-150 mm. Taking the fertilized egg in this size portion, it can be seen that the fertilized egg has the advantage that the surrounding capsule of the dark white appears to have an optimal thickness.

The incubator is a liquid white diluted with water or salt solution. It is usually preferred to start cultivation with a white solution diluted with a salt solution (eg 3: 2) and then dilute the white solution with a salt solution (eg 2: 1).

The method according to the features of the invention is preferably carried out in a closed container. The vessel is made of an impermeable material such as glass, which is sealed with a low gas permeable membrane from Saran Lab SARAN WRAP ™. Other materials with suitable properties of saran wrap can also be used. Gas permeability is measured as described above (eg carbon dioxide). The 24 hour pH rise was 0.5-1.0 in the carbon dioxide permeability test. For example, 0.6 to 0.8. The water vapor permeability is set to 1.0 to 10, for example, 2 to 5 mg / cm 2 / 24h.

Embryos cultured according to this feature of the invention are then cultured according to the second method of the invention. Therefore, according to a fifth aspect of the present invention, there is provided a method for in vitro culturing of algae embryos up to germline formation and during embryonic morphogenesis, which method comprises culturing algal embryos by the method according to the fourth aspect of the present invention. Next, the embryo is cultured by the method according to the second aspect of the present invention.

If it is desired to use a practically complete culture system of algal embryos from fertilized eggs to hatching, it can be seen that it is appropriate to apply the various features of the present invention in sequence. Therefore, according to a sixth aspect of the present invention, there is provided a method for in vitro culturing algal embryos during embryonic morphogenesis and while embryos grow and hatch until embryonic formation, which method is in accordance with the fourth aspect of the present invention. After culturing the algae embryo by the method, after culturing the pear by the method according to the second aspect of the present invention, the pear is cultured by the method according to the first aspect of the present invention.

In general, when eggs are used as containers in the second stage, the receiving eggs are slightly larger than the donating eggs, for example in the amount of 1-2 ml. If eggs are used as containers in the third stage, it is desirable that the receiving eggs be substantially larger than those used in the previous stage (eg about 18 ml).

In order to better understand the present invention and to demonstrate the effects, it will be described in detail below.

The present invention will be described with reference to various examples, in which the following materials are used unless otherwise stated.

The viability of the cultured embryos is summarized in Table 1.

animal. Hens of the usual Warrens (Isa Brown) species are kept in each cage and kept in a 14 hour light / 24 hour cycle.

At 28-32 weeks, when the eggs were laid for a long time of 1 egg / day, freshly collected semen from the Roe Island Rad Rooster was artificially fertilized.

Fertilization regularly checks uncultured eggs by visual inspection, and it can be seen that more than 90% is good. In the fertilized egg, the germinal part appears as a white ring (3-4 mm in diameter) surrounded by a translucent area and in the unfertilized egg it appears as a scarlet (2-3 mm in diameter).

egg. Eggs laid over 24 hours as the prepared species are used as a source of embryos of culture systems II and III (Examples 1 to 3). In addition, new eggs (fertilized and unfertilized) from these species are used as a source of white incubators. Liquid whites are collected from the inner and outer white layers of eggs (Figure 2) and used for incubation as the same. The receiving angle (Examples 2 to 5) of the culture system (II) is obtained from a low species. Larger angles (Examples 1, 3, 5) used as culture vessels of system (III) were obtained from fat hatcheries (D. B. Marshall, Whitman, West Rotian) and used for cultivation within 1-2 weeks of oviogenesis. Obtained from double-yolk eggs of chicks.

Lapping membrane. Two plastic wraps are used to seal the culture vessel. Saran Lab (Dow Chemical Company) has low gas permeability (Dun, Fitzharis & Barnett, 1981) and is best suited for system (I).

Mucous membranes (brands other than those lacking PVC additives) are partially permeable to gas (Don et al., 1981) and are used in systems (II) and (III). Permeability testing is done with the lapping membranes currently used with the conditions used to culture the cultured embryos. Permeability to CO ₂ was indirectly measured by measuring the pH rise of the incubator (liquid white: salt solution, 2: 1) in a glass vessel (60 ml; diameter, 40 mm) containing 25 ml of incubator and sealed with a wrapping membrane. Measure Inject the pH into the incubator to lower the pH to an appropriate value. In the Saran wrap (one layer), the pH rises on average from 0.7 to 8.2, from pH 7.2 to 7.5, during pH 24 hours at 41.5 ° C. and humidity zero. In mucosa (two layers), the pH rises from pH 7.2-7.5 to pH 8.3-8.5, on average 1.0 units during 24 hours incubation at 38 ° C. and RH 45-55%. The permeability of water vapor is measured by measuring the water lost in a dish containing 150 ml of water (350 ml; 104 mm in diameter). The dish is sealed with a wrapping film and incubated as described above. In SaranLab, the mean permeability was 3.4 mg / cm 2/24 hr (range, 3,203,8 mg).

In the mucosa, the mean permeability is 22 mg / cm 2/24 hr (range. 15-28 mg).

culture. A cabinet-type culture instrument (CURFEW Model 248) with many forced air, automatic conversion mechanisms is used for cultivation of the embryo. The conditions of temperature, humidity and inclination angle of the dish in each incubator are adjusted according to the requirements in the special period of embryo growth. The cultures are transferred from one incubator to the next, at appropriate times, and examined at intervals of two or three days or more. During and after incubation, the culture is placed on a table top, in a fixed air culture instrument (CURFEW Model 146) with a transparent lid for frequent inspection. Humidity is maintained at a given level and measured with a fish hair hygrometer, using a dish of water (2 liter capacity) at the bottom of the incubator. The machine is cleaned and sterilized monthly with MILTON sterilization fluid (Richard Sun-Viks Company).

Aseptic condition. All manipulations are done under semi-sterilization conditions. It is not necessary to take complete sterile prevention as it prevents bacterial growth on egg whites.

Eggs are usually washed briefly with 70% alcohol after collection and then wiped with 70% alcohol just before use.

All devices, distilled water and salt solutions are pressurized with an autoclave. The salt solution is filtered and sterilized. In the lapping film, the outer layer of the roll of the film is discarded, and then the sheet (100 mm 2) is cut and placed between the sterilizing papers. This operation, collection of egg whites and preparation of the culture are performed in an air cabinet with a lock.

Antibiotic (penicillin, 100 ml) is added to the egg white and glued to the mucosa to seal the receiving angle of system (III).

Example 1

Embryo culture, culture system (III) method from 4 days of growth to incubation

The receiving angle is made of double egg white, a hole of 40 mm diameter is drilled around the blunt end of the egg, and the angle cap with the air chamber is removed. After discarding the contents, each was washed with distilled water and then refilled with water to prevent dehydration of the inner cornea. The volume of water response angle is 65 ~ 75ml.

In steps 15-20, eggs are incubated for 3 days, containing pears, and the contents are lowered into a shallow dish at the mucosal level. The abdomen is moved from the mucosa sac to the receiving angle, and then slowly withdraws the mucosa while keeping the abdomen at its best. Detailed previous methods have been reported by Lloret and Simkis (1987).

The liquid is enclosed in two layers of mucosa using a liquid white glue glued to the membrane. The space depth between the vessel and the membrane is 10 mm on average. Cultures are incubated at 38 ° C. and shaken intermittently at an angle of 30 ° for 5 days and then held in a floating position for 10 days. For the last 3-4 days, they are placed in a floating incubation apparatus at 37 ° C. Relative humidity is 45-60%. One or two days before the hatching time, a small perforation is made in the mucosa as the beak penetrates the intestinal membrane and penetrates the space. The mucosa is replaced with a Petri dish cover several hours before hatching.

The hatching rate was 36% (Table 2) and the healthy hatching was 72%.

Their average weight was 35 compared to 46 of the comparative chicks bred previously. Abnormal weakness includes incompletely shrunken yolk sacs, unrecovered centers and limbs. Sticky chicks are common, and the conditions are associated with the presence of some unabsorbed whites at each.

^{+ The number of} hatched chicks includes healthy chicks and weak chicks. Ono and Wakasugi (1984) refer to each technique for cultivating quail embryos obtained from eggs incubated for three days; They demonstrate that egg shells are an essential source of calcium in the ship. Lloret and Simkis (1985, 1987) adapted the technique to chickens and hatched at 20%. The function of the intestinal membrane in calcium absorption and gas exchange can be seen that the ball is not damaged by seed or species differences in the vessel and plinth.

Rotation of the cultures during the first incubation was mentioned by Lloret and Simkis (1987). Rotating the hen's eggs during the entire incubation period is a common practice, but New 1957 indicates that the critical period is between three and eight days of incubation. Switching cultures in a series of tests is required for optimal growth and this exercise appears to be necessary only during the first 5 days of culture (4-9 days of growth). Experience showed that the unconverted culture had an incubation rate of 18% (n = 77) as compared to the 36% hatching rate (Table 2) of the culture rotated for 5 days, at which time it was maintained at a stationary position. Extending the rotation period to 15 days results in a worse hatching rate. As a percentage of survival fold on day 9 of growth, hatching rate was 62% (n = 33) for cultures rotated during the first 5 days and 40% (n = 71) for cultures rotated for 15 days.

Receiving angle mucosa seals are used in place of flow covers used by other operators. The advantage of this modification is that by using the standard conditions of egg culture (forced air system, RH 50-60%), the intestinal mucosa region associated with the receiving angle receives a normal environment. At high humidity above RH70%, the hatching rate is reduced by 10% (n = 38). The mucosa is effective in controlling the environment of the space above the ship by limiting gas exchange and water lost in these areas. In the egg chamber, the water vapor pressure is higher than ambient and the O ₂ tension drops as CO ₂ tension rises with incubation time (Wanggensteen and Ron, 1970/71). Wrapping membranes that are partially permeable to gas have been shown to provide optimal conditions for chick embryos to develop in angular cultures. (Dun et al., 1981). In the non-bottom angle experiments, the cultures are incubated at high humidity in air for chicks (Lowert and Simkis, 1987) with 1.5% CO ₂ for quails (Owawa Kasugi, 1984). The latter study indicates that maintaining the culture of air and other O ₂ / CO _{2 in} the vessel is of less importance for normal growth of the culture than to prevent water loss from the unprotected intestinal membrane facing the chamber. In this example, the bilayer of the mucosa was used with some environment of several ships that survived 19 days at an angle that was underpinned by a monolayer. The second advantage of the mucosa is to help reduce contamination by microorganisms.

Example 2

Culture of embryo from 1 to 9, culture system (II) method

Eggs 3 to 4g heavier than the donor eggs were chosen as acceptance angles. A 32mm diameter aperture drills the tip and angle and discards the contents. Each is washed with distilled water and then refilled to prevent dehydration of the inner cornea. The angle of the uncultured, fertilized egg is cut with scissors, the cracked angle is opened by hand, and the contents are dropped into a glass bowl (diameter, 50 mm; volume 60 ml). These are transferred to the receiving angle through the Bikini (diameter, 35 mm; volume 70 ml). In this process, minimal damage to the capsules of the array, yolks and viscous whites was identified. Fill the edges of the bowl with the selected liquid whites (1-5 ml) from the newly laid eggs and seal the holes with mucosa sheets, taking care not to create bubbles during preparation. The mucosa is held in place with a two-ring (nylon) at the tip of the angle and secured with an elastic band hung over a set of small pegs. The prepared eggs are incubated on their side and shaken intermittently or continuously at a 90 ° angle in time cycles at 38 ° C. and 30-50% relative humidity. Lighten the cultures on fire at day 7.9 and day 10 and reculture the viable embryos. Embryos showing normal growth were observed at 74% of the culture on day 7 of culture (Table 3). Mortality was high after two days and only one ship survived after 10 days. At all stages of preparation, air chambers are inflated to replace the water lost by evaporation.

Kalebaut (1983) described a method of cultivating quail pears in each egg for 2-3 days. (Data did not hesitate). This method consists in transferring the contents of the sterilized donor eggs to the empty main angle and then sealing the formulation with a Petri dish and applied paraffin wax. In chick embryos, the method seals and rotates the formulation during incubation to purify using the mucosa. In stationary culture experiments the survival rate is 50% at 7 days and these embryos are usually supported on growth. In culturing the 1-fold fold in culture systems (I) and (II) containing gas chambers, it can be seen that mortality is high for 2 to 3 days of culture. Similar observations were made by Dun et al (1981) in their angular cultures. The failure of growth in these crabs is due to the fact that the germ is only protected by a thin layer of white egg. Romernov (1943) emphasized the importance of liquid whites in the growth of germ layers in whole yolks. Continued fluid, ventilated angles of the germ layers with liquid whites assist in the formation of subventral cavities. These cavities are filled with fluid derived from the whites at initial growth. This process also indicates a preparation that lacks a sufficient amount of white near the belly. Carl Bowt's (1983) culture system of quail embryos was modified in this example by rotating the culture at the time of incubation, adapting it to chick embryos, or replacing the mucosa of the Petri dish / paraffin wax seal, among other things. Fertilization expanded growth by 4 days of culture and resulted in high survival rates.

Example 3

Culture culture systems (II) to (III) of embryos from day 1 to incubation

Embryos are incubated for 3 days as described in Example 2 in uncultured eggs. (Figure 4). The agent is removed from the culture apparatus at once, then the angle is cut and the contents transferred to the larger angle as described in Example 1 (Figure 3). The removal of smaller angle contents through the openings in them always damages the abdomen and outer membrane. Cultures are cultured as described in Example 1. Survival rates are shown in Table 4.

+ The number of hatching chicks includes healthy birds and weak birds. In the early part of the culture, losses have been mixed with embryos that failed to grow in system (II) and damaged embryos during transfer to system (III). The mortality rate between three and nine days depends on the damage during the transfer. The hatching rate was 27%, and as described in Example 1, about 20% of hatching showed leg and yolk sac defects. Several pups were raised and tested for their spouse's viability. The hatching rates (89% and 75% egg production, respectively) of the two experimental hens artificially fertilized from the Roeland Rad Rooster were 63% and 80%, respectively. Egg hatching rates were 80% and 15%, respectively, in inferior hens fertilized from two experimental roosters. Both experimental hens did not lay eggs.

In culture system (II), growth arrest occurred at 8-10 days of growth and was shown to be associated with a lack of contact between the intestinal and internal corneas. Splitting of egg yolks in manufactured eggs affects the buoyancy of the yolk and also damages the mechanism of gas exchange by the distance between the cornea and the outer-membrane. To solve this problem, the ship is transferred to a system near the air / white boundary of the outer-wall of the conduit. It was at the end of three days of incubation when the growth of the time temperature conduit best suited to transfer to the culture system was achieved and the components of the manufactured eggs were strong enough to withstand handling. In some experiments (figure 3) in which embryos were cultured from day 1 of growth at a stop angle with air chambers, survival was low and hatching capacity was zero. (M. age, personal communication). Linking the phase (III) system from phase (II) is a new process. This is a report of successful cultivation of algal embryos from embryonic stage (step 1) to hatching.

Example 4

Fertilized egg culture up to 7 days, culture system (I) to (II) method

The hens were examined at 2.75 hours after evaluating oviparum which confirmed fertilization in live eggs. During this daily regeneration cycle the next egg is ovulated and the whites settle and cross. In order to recover the egg tube, the hens are killed by intravenous pentobarbitone sodium, and the hair of the abdomen is pulled out and washed with 70% alcohol. A portion of the oviduct with fertilized eggs is lifted out of the abdominal cavity, cut and placed in a sterile bowl with paper soaked with salt. Most eggs are located 50-150 mm from the edge of the canal size. Make a long incision in the wall of the oviduct and place the egg in a glass beaker (diameter, 35 mm; volume 70 ml) while avoiding damage to the viscous white capsule around the yolk. The eggs are transferred to a glass vessel (diameter, 40 mm, volume 60 ml) containing about 5 ml of incubator and the yolk is moved to the best placenta. The incubator is added at the level of the blastocyst, below the white capsule surface, and the container sealed with Saran wrap is secured with a stretch band (FIG. 5). The total volume of incubator required depends on the size of the yolk and white capsules and the amount is 8-12 ml. In cultures in which the betrayal is placed on the yolk, the vessel is inclined so that the white capsule on the betrayal is not submerged in the incubator. The formulation is delayed to 20 minutes or less between the size modification and incubation of the eggs and incubated at 41-42 ° C. for 24 hours. The incubator consists of liquid white (2 parts) and salt solution (1 part) collected from the inner and outer white layers of newly laid eggs. The salt solution contains 50 mM KHCO ₃ , 30 mM NaHCO ₃ , 10 mM KCl, 2.5 mM MgCl ₂ · H ₂ O, 0.7 mM CaCl ₂ · 2H ₂ O and 11 mM glucose. The pH of the incubator is lowered from the initial value of 8.4 by stirring in CO ₂ gas to 7.2 to 7.4, and the incubator is stored in a sealed container and kept at a low value during the manufacturing process. After 24 hours, the growth status can be confirmed completely by visual examination of the germination area. Embryonic growth and subventricular cavity formation are indicated by opaque rings (3 mm in diameter) stacked semi-transparent portions. However, to accurately assess growth potential, the preparation is incubated for three to six more days in system (II). Embryos are placed in phase II culture system as described in Example 2. Acceptance angles are made from eggs about 3 to 4 g heavier than the eggs described above by the donor hen. The culture is cooled to room temperature, placed in a receiving angle filled with the incubator, and sealed with mucosa (Figure 4). Prepared eggs are incubated at 38 ° C RH 30-50% while shaking intermittently at a 90 ° angle at time intervals. Cultures are removed from each to be examined on culture 7.8 or 9 days. The results are shown in Table 5 below.

Normal embryos grow in steps 27-29 in 67% of the cultures. They usually die at the beginning of 8 days of culture and do not survive outside this stage. In the remaining cultures, cells grow or do not grow, or there is a doubling of malformations (see Tables 6 and 7).

During the first 24 hours, the reorganized eggs are placed in a sealed plastic bag and shaken at a 90 ° angle in a time period. Next, these are cultured by the method of system ().

The growth period is delayed by 12 hours or more.

+ Mortality was high in 7-day cultures in undiluted whites and 8-day cultures in diluted whites.

In these formulations, the culture vessel for phase (I) is sealed with mucosa and the mucosa layer is placed directly on the white capsule.

The phase (I) culture system is based on a series of experiments designed to test the requirements for the first 24 hours of growth.

Testing is done with eggs at different stages after sterilization and with different treatments prior to transfer to a standard (II) culture system for different growth and analysis. The characteristics of the cultures examined were: depth of material across the betrayal, composition of the incubator, gas exchange and spatial location of the betrayal.

An important factor is the depth of the whites on the stomach. Eggs stacked with relatively thin white capsules in large front varieties or thick white capsules in large rear varieties have lower survival than fertilized eggs with medium-sized varieties (50-150 mm in buccal). Removing the egg white capsules causes the betrayal to float on the surface of the incubator, preventing growth in medium-sized varieties.

In addition, growth is inhibited when the capsule completes the capsule in the incubator. Most of the failure causes of growth in the experiment (Table 6) on the use of the culture system (II) (FIG. 4) of the sterilized eggs are due to the precipitation of the encapsulated eggs in the incubator. In these formulations, the first 24 hours are incubated with the best sealed surface and the distance of the betrayal in the mucosal seal changes with the buoyancy.

The incubator described above is not essential for growth in phase (I).

Pears grow well in undiluted whites for 6 days in stages 25-26. However, growth beyond this stage requires the use of diluted whites (Table 7). The composition of the salt solution was based on the data provided by Bidle, Conrad and Scott (1983) and Reward (1968) for the difference in the ionic composition of the egg white of the uterine egg and the egg white of the laid egg before expansion. Glucose is added at the same concentration as the uterine secretion (Devison's gland and Drapie, 1969) at the onset of uterine formation of egg formation. In reorganized eggs, the total amount of secretion (salt solution and swelling secretion) is completely similar to the amount of swelling secretion in the laid eggs. The swelling secretion consists of about half of the egg white content, which varies between 36 and 40 ml between days. Experiments in which the whites of the incubator were diluted with distilled water only showed similar results as using a salt solution as a diluent (Table 7).

Thus, for the short term incubation with step 29, water is an important component.

The balance of the gas atmosphere with the associated acid-base of the incubator in the culture vessel is not necessary to adjust the actual growth in the ophthalmic deposits.

The pH of the egg white in the uterus varies from 7.15 to 7.4 (Hover and Mongin, 1971). Due to the CO ₂ outflow from egg to egg, the pH rises to 8.4 within a few hours of birth (Dauth, 1975). In addition, the oxygen content of the egg white in the uterus is low, which is in equilibrium with air within 2 hours of incubation in eggs (Wishaart, personal communication). In the phase I culture method, the pH of the incubator is adjusted to 7.2-7.4 with CO ₂ to comply with in vitro conditions; Oxygen levels in the incubator are as close to ambient

The vessel is sealed with a saran wrap to maintain a pH of 7.8 or less and a damp atmosphere in the room.

There is a slight decrease in the number of viable embryos at higher pH, but no growth is seen by culture. The fertilized egg is placed at the receiving angle and filled with an incubator at pH 7.4 to the level of the line covered with the betrayal and cover. They are incubated at 42 ° C. for 24 h with RH 80% in 10% CO ₂ or air. The pH of the incubator is raised to an average of 0.31 units and 1.15 units, respectively. The next embryo is incubated in further system (II) for 6 days. Normal embryos grow in 55% (n = 33) of culture in 10% CO _{2 and} in 41% (n = 29) of culture in air.

It is proposed to measure bilateral symmetry on the uterus of growth by specific gravity (Kochou and Iyal-Guiladi. 1971). Experiments are measured at spatial locations in the germination zone. During the critical period, the embryonic axis forms an inclined vessel and is not positioned horizontally (Uljaska, Shorazuka and Lasota, 1984).

There was no evidence to support them in this work.

Extussis is formed in 68% (n = 34) cultures grown in horizontal ovulation and 74% (n = 34) cultures in inclined blastocysts.

The combination of the method of phase (I) with the method of phase (II) is completely new. This is the first report of algal embryos growing in vitro from fertilized eggs to stage 29 during a third of embryo survival. There are two different reports on the culture of the oviduct in the whole yolk. Howard (1971) incubated the eggs obtained in the front region of the oviduct in an alignment step (step 1) with a survival rate of 60% (n = 10). To prevent the yellowing from floating on the surface, at this stage, eggs that lack white capsules are placed in a plastic angle dipped in a beaker of liquid white. Cochow and Iyal-Guiladi (1971) grew uterine embryos from the multicellular stage to the 4-harvest stage (step 8). In this study, the cornea was removed, the yolks and egg whites were transferred to a beaker of pharmacological saline solution, and the yolks were lengthened into ovaries, making the embryonic area inclined.

Cultivation methods for the growth of fertilized eggs during the 7-day period provide an exemplary system for testing the effects of experimental coordination with preliminary cell division in the growth of birds. In this experiment, exogenous genes are injected into the embryo's cytoplasm and examined for embryo loss at intervals of two to seven days.

Example 5

Incubate until fertilization of fertilized eggs. Culture system (I) to (II) to (III) method

In the medium-sized varieties of the oviduct, the rare eggs are incubated for 24 hours in the phase (I) system (Figure 5), transferred to the phase (II) system (Figure 4) and incubated for 3 days. This process is described in Example 4, and the ratio of the liquid white and the salt solution in the incubator was adjusted to 3: 2 for the system (I) and 2: 1 for the system (II). To ensure that the embryo is supplied with the required amount of incubator for long-term growth, the receiving angle produced for system (II) is 1 to 2 ml larger than the above-mentioned eggs laid by the donor hen. After a total of 4 days of incubation, the embryos are transferred to the phase (III) (FIG. 3) system as described in Example 3. The receiving angle produced for the system (III) is 18 ml larger on average than the receiving angle used for the system (II). Volume differences were measured in room size in yarn. Incubate at 38 ° C. for 5 days at 30 ° C., then incubate for 10 days at a stationary position, and finally put in a stationary hatching culture apparatus at 36-37 ° C. (Table 8).

The results of Experiment 9 in which the cultures were incubated for 4 to 19 days at R. H. 30-55% relative humidity, are shown in Table 9.

⁺ Five chicks are healthy

Initial loss is the sum of embryos that failed to grow on day 4 and embryos that were damaged during the transition from system (II) to (III) on day 10. Embryos cultured in fertilized eggs are biliary and are more sensitive to damage than embryos cultured from day 1. The hatching rate is 8% and about 8% of the hatches are healthy. At high humidity, R. H. 50-60%, hatching rates were similar (n = 85), but a single hatching was healthy.

One weak chick hatched in culture was incubated in R. H. 60-75%.

In total, seven healthy chicks were hatched from cultured fertilized eggs, including those cultured by a modified method. The two roosters lived and matured: one fertilized with a 75% hatching rate from fertilized Wazen hen eggs, the other unfertilized. Nine weeks later, the two hens were healthy. The remaining birds lived for 1, 8 and 16 weeks, respectively.

The reasons for adopting three separate cultures during successive growth periods are described above. The upper layer plays a decisive role in the growth of the embryo. The proper correlation of the embryo with the upper layer is measured empirically, by adjusting the design of the culture chamber appropriately. Change requirements in different phases of growth. In phase (I), excess incubator of embryonic phase is detrimental, and in phase (II), this is essential for growth. When growth is not inhibited unless the outer-membrane of the catheter is exposed to the atmosphere in the chamber, the excess incubator is 8 It is not harmful in phase (III) until day.

The combination of systems (I) to (II) to (III) to maintain the growth of chick embryos in culture in a short step until fertilization after fertilization is a new method in itself. This is the first report of a complete culture of algal embryos (Ferry, 1988).

The establishment of a complete test relationship for chick embryos has a wide range of potential uses in basic and applied research.

This gives the opportunity to adjust the new eggs, for example by injecting other genes or whole genomes, or to study the effects of such adjustments in hatched chicks and in mature age. This method is also a guideline for creating in vitro techniques for oviducts in oviducts at advanced stages faster than currently in use, by using the field of in vitro fertilization and inserting putative very potent cells into the embryo.

Potential use is in making poultry from previous genes. Changes in factors for growth and reproduction completion will benefit the poultry industry. Moreover, incorporating the new protein's genetic factors into the algal germline is a potentially valuable technique for making biomedical proteins from white eggs. The high reproduction power of poultry also benefits other livestock in the art. Hens mature to six months and can lay about 300 eggs in their first year of laying.

In summary, isolated cultures consist of three phases of growth, and embryos are transferred from one to the next to cover the entire embryo life. The order of all experimental designs and methods is shown in Figure 1 in an urban form. An exemplary method for phase (III) is a separate system (Example 1). An exemplary method for phase (II) spans that for phase (III) (Example 2).

However, the transfer between these systems is preferably carried out at special stages due to the increased fragility of old ships (Example 3).

The method of phase (I) is covered in a period starting about 2 hours after ovulation, when the egg white is deposited down and the sperm nucleus before fertilization of the male and female is expanded (Ferry, 1987). Here a combination of the method of phase (II) for analysis (Example 4) and the method of phase (III) for complete culture system (Example 5) is required.

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Claims (15)

In vitro culture of chick embryos in embryonic growth, characterized in that the vessels are cultured in a closed container and the vessels have air chambers, and the chambers are separated from the outside atmosphere by a seal through which gas can partially permeate. The culture method according to claim 1, wherein the container is part of an egg. The culture method according to claim 2, wherein the eggs are eggs of hens and the space depth between the belly and the seal is 5 to 15 mm. The culture method according to claim 1, wherein the cultured embryos are gently stirred at least initially. Chick test tube culture method characterized in that the embryo is cultured in an incubator in a liquid-filled sealed liquid-impermeable container. 6. The culture method according to claim 5, wherein the vessel is partially permeated with gas. The culture method according to claim 5, wherein the incubator is liquid white. 6. The culture method according to claim 5, wherein the cultured embryos are subjected to gentle moderate agitation. An in vitro culture method of a chick embryo on embryo growth during embryo development, characterized in that the embryo is first cultured by the culture method of claim 5 and then the embryo is cultured by the culture method of claim 1. In vitro culture of chick embryos up to germ formation, characterized by culturing fertilized eggs, with surrounding capsules of dark white, partially precipitated in the incubator. The culture method according to claim 10, wherein the incubator is in the line with the betrayal. The culture method according to claim 10, wherein the incubator is a liquid white diluted with water and a salt solution. The culture method according to claim 10, wherein the culture is carried out in a sealed container. An in vitro culture of algae embryos until embryonic formation and embryonic development, characterized in that the chick embryo is cultured by the culture method of claim 10 and then cultured by the culture method of claim 5. After cultivating the chick embryos by the culture method of claim 10, the embryos are cultured by the culture method of claim 5, and then the embryos are cultured by the culture method of claim 1 to the embryonic morphology, embryo growth until embryonic development and hatching In vitro culture of chick embryos.

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